Jasmonates are essential modulators of plant defences but the role of JA-derivatives has been scarcely studied, particularly in the plant-pest interplay. To deepen into the JA catabolism and its impact on plant responses to spider mite infestation, we selected the Arabidopsis JAO2 gene as a key element involved in the first step of the JA-catabolic route. JAO2 is responsible for the hydroxylation of JA into 12-OH-JA, contributes to attenuate JA and JA-Ile content and consequently, determines the formation of other JA-catabolites. JAO2 was up-regulated in Arabidopsis by mite infestation. Mites also induced JA-derivative accumulation in plants. In jao2 mutant lines, and in the triple mutant jaoT (jao2-1, jao3-1, jao4-2), mite feeding produced less leaf damage, minor callose deposition and lower mite fecundity rates than in Col-0 plants. The impairment of JA oxidation in jao2 lines not only diminished the 12-OH-JA levels but turned off further sulfation as shown the significant reduction of 12-HSO4-JA form. Thus, JAO2 acts as a negative modulator of defences to spider mites mediated by changes in the generation of JA catabolic molecules, and the consequent production of defensive metabolites such as glucosinolates or camalexin.